The Gomoris methanamine silver impregnation technique is a highly reliable and archiveable method of detecting fungal filaments, but the staining procedure is time consuming and laborious. A technique using microwave energy to reduce the duration of Gomori's silver staining is described.

Mycotic keratitis accounts for 17-47% of total corneal infections all over the world and is highly prevalent in southern India.[1][2] Fungal keratitis is diagnosed by detection of fungal filaments in the smears of corneal scrapings by Gram's, Giemsa and Potassium hydroxide (KOH)/calcofluor white preparation (CFW). The sensitivity of these tests ranges from 33-95%.[3],[4] The potassium hydroxide wet mount preparation and calcofluor white preparations are useful. They are more sensitive (85-92%) and cost effective, but they are temporary preparations and the latter requires a fluorescence microscope.[5],[6] The fungal stain routinely used by cytologists and histopathologists is the silver impregnation technique. This technique is highly reliable and archivable, but is time consuming and laborious.[7] We used microwave heating to reduce the time taken by the silver impregnation technique[8] and evaluated the utility of this method in comparision with the standard microbiological stains. To the best of our knowledge, backed by a literature search (Medline), this technique has not been used to diagnose corneal infections. We believe this has great clinical utility in the management of corneal infections.

Materials and methods

All patients with corneal infections attending the Cornea services at the L V Prasad Eye Institute, Hyderabad underwent a thorough clinical and microbiological evaluation as reported by us earlier.[6] As a matter of routine the provisional report on Gram's, Giemsa, KOH/CFW preparations is given within 30 minutes, while the interim culture and sensitivity report is issued within 48 hours and the final report in 1-2 weeks.

In this study we included the consecutive Gram's and Giemsa stained smears that were interpreted by the microbiologist. The same smears (without destaining), were used for Grocott's Methenamine (GMS) silver staining by the microwave heating technique using clean glassware [Table - 1]. The sections were exposed to microwave heating with "working" methenamine solution for 3 minutes at 30% power (low medium scale). The sections were then allowed to stand till they turned brown. The procedure for microwave-assisted GMS staining took about 20 minutes. The GMS smears were then interpreted by 2 independent masked observers. Considering the final culture report as gold standard, the sensitivity and specificity values were calculated and the sensitivity rates were compared using Yale's Chi-square test.

Results

Microwave-assisted GMS staining was done on 40 smears, that is, 20 Giemsa stained, and 20 Gram's-stained smears from 20 consecutive patients with a clinical diagnosis of infectious keratitis. The final culture report of these 20 cases revealed fungus in 13 and bacteria in the remaining 7. In the culture-proven cases, fungus was detected in 69.2% (9/13) cases by both Giemsa- and Gram's-stained smears. The KOH and CFW wet mount preparation identified fungus in 84.6% (11/13) cases.

The same Gram's and Giemsa-stained smears when restained by GMS revealed fungus in all 13 cases. The cellularity and morphology of cells after restaining was well preserved (Figure). The fungi were seen as prominent black filaments of varying length with two parallel borders. Septae and branching fungal filaments were clearly identified in the smears.

Inter-observer variability was good in the smear reports. The time taken for reporting varied from 20 seconds (immediate diagnosis) to 2 minutes. The sensitivity of the Giemsa and Gram's-stained smears as compared to culture results was 69.2%, with 100% specificity in both procedures. The sensitivity of KOH/ CFW preparation was 84.6%, and the GMS-stained smears was 100%. False positive report of fungus was not made in any of these preparations. The statistical significance of the sensitivity of Gram's, Giemsa and KOH/CFW with GMS staining was 0.1030 and 0.4617 respectively (Yates chi-square).

Discussion

Keratomycosis is a suppurative, ulcerative, sight threatening infection of the cornea accounting for 17-36% of corneal infections.[1][2] Despite advances in medical treatment, fungal keratitis could require surgical intervention like keratoplasty or evisceration in one-third of cases. The routinely used Gram's and Giemsa stains, KOH, and CFW preparation for fungal filaments give reliable and rapid results with a sensitivity of 35-95%.[3],[4] We, therefore considered the silver impregnation technique of detecting fungus, which is routinely used in cytology and histopathology practice. In order to reduce the time of staining, we applied domestic model microwave heating.

The staining time was reduced from the routine 3 hours to 20 minutes without compromising the quality of the staining. The fungal filaments appeared black against the green background facilitating rapid identification under low magnification, in contrast to the variable staining of Gram's or Giemsa stained smears. Furthermore, the sensitivity of Gram's and Giemsa stains in this study was 69%, slightly lower than the 86% reported by others.[4],[8]

The sensitivity of this technique is comparable to the KOH mount and CFW preparation - 84.6% in this series, comparable to the 81.2% and 93.7%, reported by Sharma et al.[6] These two techniques though sensitive, with a specificity ranging from 83.8% to 100%, cannot be preserved for longer periods. In addition, CFW staining, regarded as one of the most sensitive methods for identification of fungal filaments on corneal scrapings, requires a fluorescence microscope. This may not be affordable by all the diagnostic laboratories.

In this study, the sensitivity of GMS staining was 100% and equivalent to the results of culture, the accepted gold standard. Though the differences in sensitivity rates of Gram's, Giemsa and KOH/CFW when compared to GMS stain were statistically not significant in this study due to the small sample size, it appears to be of clinical significance. It could be applied at centres that do not have a fluorescence microscope and also in cases where other staining methods yields doubtful results. That this technique facilitates restaining and reevaluation of Gram's or Giemsa stained smears without loss of material or structural details, is an additional advantage. The precautions to be taken for this procedure are the use of clean and dry jars, and complete adherence to the protocol so as to avoid excess heating or drying of the smears. The sections could get lifted during the procedure and there could be stain deposits and unequal staining due to unequal heat distribution. These problems can be overcome by standardising the time according the glassware and the type of microwave used. To minimise the risk of exposure of the technical staff to fumes, the microwave should be kept in the laminar flow hood.

In summary, rapid detection of fungal filaments on corneal scrapings can be facilitated by microwave-assisted GMS staining, which is a highly sensitive and specific method of detecting fungus. This would greatly help clinicians arrive at an accurate diagnosis of fungal keratitis and initiate early treatment with antifungal therapy. This could be of significant value especially in centres that lack a fluorescence microscope.